Emerging infections by orthopoxviruses and their potential use of bio-weapons has generated an urgency to better understand factors controlling poxviral pathogenesis. Our ultimate goal is therefore to characterize the molecular function of important virulence factors of orthopoxviruses thus providing novel strategies for therapeutic intervention and vaccine design. Poxviral virulence is directly correlated with the ability of viruses to overcome innate and adaptive host responses. The highly conserved poxvirus protein P28 is an important virulence factor and seems to be essential for growth in macrophages. The molecular function of P28 is unknown but evidence exists that it protects against apoptosis. The first specific aim of this application is to test the hypothesis that P28 protects macrophages from virally induced apoptosis. By introducing P28 into the vaccinia virus strain W811 that lacks other viral modulators of apoptotic pathways we will further characterize the role of P28 in preventing apoptosis induced by either viral infection or external stimuli. The second specific aim is based on our preliminary observation that P28 represents the only known orthopoxviral ubiquitin ligase of the RING-E3 type. This function implies that P28 targets other proteins, either viral or cellular, for ubiquitination. We will use two proteomics approaches to identify such targets as well as other changes in the host cell proteome mediated by the ubiquitin ligase function of P28. Differential two dimensional gel electrophoresis and tandem-mass-spectrometry will be used to identify changes in the proteome of cells infected with vaccinia virus expressing wild type or RING-mutant P28. As a complementary approach we will use sequential liquid chromatography and tandem-MS to compare the ubiquitinated proteome. The results of the proteomics analysis are likely to provide novel insights into the molecular function of P28 which might uncover novel cellular or viral pathways modulated by P28. How the molecular targets of P28 are involved in aiding the virus to grow in macrophages will become an important question that will be addressed in future studies based on this exploratory application. In addition, the proteomics approaches are expected to provide new tools for the study of ubiquitination events in virally infected cells.